Genome reduction in novel, obligately methyl-reducing Methanosarcinales isolated from arthropod guts (Methanolapillus gen. nov. and Methanimicrococcus)

Abstract Recent metagenomic studies have identified numerous lineages of hydrogen-dependent, obligately methyl-reducing methanogens. Yet, only a few representatives have been isolated in pure culture. Here, we describe six new species with this capability in the family Methanosarcinaceae (order Methanosarcinales), which makes up a substantial fraction of the methanogenic community in arthropod guts. Phylogenomic analysis placed the isolates from cockroach hindguts into the genus Methanimicrococcus (M. hacksteinii, M. hongohii, and M. stummii) and the isolates from millipede hindguts into a new genus, Methanolapillus (M. africanus, M. millepedarum, and M. ohkumae). Members of this intestinal clade, which includes also uncultured representatives from termites and vertebrates, have substantially smaller genomes (1.6–2.2 Mbp) than other Methanosarcinales. Genome reduction was accompanied by the loss of the upper part of the Wood–Ljungdahl pathway, several energy-converting membrane complexes (Fpo, Ech, and Rnf), and various biosynthetic pathways. However, genes involved in the protection against reactive oxygen species (catalase and superoxide reductase) were conserved in all genomes, including cytochrome bd (CydAB), a high-affinity terminal oxidase that may confer the capacity for microaerobic respiration. Since host-associated Methanosarcinales are nested within omnivorous lineages, we conclude that the specialization on methyl groups is an adaptation to the intestinal environment.


Introduction
Evidence for the presence of obligately methyl-reducing methanogens in many so-far uncultured archaeal lineages has sparked debate about the implications for the evolutionary origin of methanogenesis (Borrel et al. 2019, Wang et al. 2021, Mei et al. 2023 ).Ho w e v er, onl y six species with this capability have been isolated, which limits biochemical and physiological experiments to elucidate their metabolic features .T he isolates fall into four distantly related orders and belong to the genera Methanosphaera (order Methanobacteriales ), Methanomassiliicoccus and Methanomethylophilus (order Methanomassiliicoccales ), Methanonatronar chaeum (order Methanonatronar chaeales ), and Methanimicrococcus (order Methanosarcinales ).
Members of the order Methanosarcinales are morphologically, bioc hemicall y, and genomicall y distinct fr om other methanogens and occur in a wide range of en vironments , such as marine sediments , wetlands , soils , and the intestinal tracts of animals .T hey show the widest substrate range of all methanogens, including not only the classical substrates CO 2 , methanol, and acetate but also methylated sulfur compounds and methoxylated aromatic compounds (Kurth et al. 2020 ).In contrast to other methanogens, Methanosarcinales possess cytoc hr omes and membrane-associated electr on tr ansport c hains, r esulting in the highest gr owth yields among methanogens (Thauer et al. 2008 , Mand andMetcalf 2019 ).
The only cultured re presentati ve of Methanosar cinales from animal guts is Methanimicrococcus blatticola , isolated from the hindgut of the coc kr oac h P eriplaneta americana (Spr enger et al. 2000 ).It differs from other members of Methanosarcinales in its restriction to methanol or methylamines as methanogenic substrates, an obligate dependency on external H 2 resulting from the inability to oxidize methyl groups to CO 2 , and a r equir ement for se v er al gr owth supplements including acetate and coenzyme M (CoM).Comparative genome analysis revealed that M. blatticola has a highly reduced genome and lacks essential elements of the methanogenic pathways present in other Methanosarcinales , including the Wood-Ljungdahl pathway and many membrane-associated complexes (Thomas et al. 2021 ), which explains the limited substrate spectrum and growth requirements documented in earlier studies (Sprenger et al. 2000(Sprenger et al. , 2005 ) ).
Methanimicrococcus -r elated sequences hav e also been detected in 16S rRNA-based studies of other coc kr oac hes, termites (the closest r elativ es of coc kr oac hes), and scar ab beetle larv ae (Ohkuma et al. 1999, Friedrich et al. 2001, Egert et al. 2003 ), and mor e r ecentl y also in millipedes (Protasov et al. 2023 ).Together with sequences from vertebrate guts, they form a monophyletic clade that consists exclusiv el y of r epr esentativ es fr om the intestinal tract of animals (Thomas et al. 2022, Protasov et al. 2023 ).
Her e, we r eport the isolation and physiological c har acterization of six new species of obligately methyl-reducing methanogens from the hindgut of coc kr oac hes and millipedes.Based on a previous, phylogenomic analysis of archaeal diversity in arthropods, whic h cov er ed both isolates and uncultur ed linea ges, the isolates wer e alr eady described under the Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode) (Hedlund et al. 2022 ) as new species of the genera Methanimicrococcus and Methanolapillus (Protasov et al. 2023 ).In the present study, we provide a detailed c har acterization of these taxa and their formal description under the International Code of Nomenclature of Prokaryotes (ICNP) (Oren et al. 2023 ), together with a compar ativ e anal ysis of all available genomes of host-associated Methanosarcinales that includes uncultur ed r epr esentativ es fr om termites and v ertebr ates.

Enrichment and isolation
Coc kr oac hes ( Arc himandrita tessellata , Eublaberus serranus , and Henschoutedenia flexivitta ) and millipedes ( Atopochetus caudulanus , Archispirostreptus gigas , and Anadenobolus monilicornis ) were obtained from commercial breeders (Jörg Bernhardt, Glashütte, Ger-man y, www.sc haben-spinnen.de;Home of Insects, Erfurt, Germany, www.home-of-insects.com ) and maintained in our laboratory as previously described (Schauer et al. 2012 ).Adult specimens were dissected, and the entire gut was placed in a sterile Hungate tube containing 2-mm glass beads (2 g).After addition of 10 ml AM7 medium, the tube was closed with a rubber stopper, the headspace was gassed with N 2 /CO 2 (80:20), and the gut was homogenized b y v ortexing the tube for 2-5 min.The gut homogenate was inoculated into culture tubes containing 4.5 ml of AM7 medium with methanol (50 mM) under a headspace of H 2 /CO 2 (80/20) and incubated at 30 • C. Cultures that produced methane wer e tr ansferr ed into AM7 medium with 50 mM methanol supplemented with kanamycin and ampicillin (each 100 μg/ml final concentr ation).Pur e cultur es wer e obtained by the isolation of individual colonies from deep-agar dilution series with 1% agar (Pfennig and Trüper 1981 ) using the same medium and growth conditions .T he absence of bacterial contaminants was assessed by inoculating the cultures into AM7 medium with 10 mM glucose.Methanimicrococcus blatticola (DSM 13328) was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Br aunsc hweig, German y).

Growth, medium requirements, and physiology
Gro wth w as measured directly in the culture tubes by following the increase in optical density at 578 nm (OD578) using a culture tube photometer (Spectronic 20 + , Milton Roy; path length ca.1.3 cm).Dry weight was determined with triplicate cultures grown on methanol (50 mM) in 1-l glass vessels containing 500 ml AM7 medium under H 2 /CO 2 .After OD measur ement, the cells wer e harvested by centrifugation (10 000 × g ; 20 min), washed with ammonium acetate solution (20 mM), and dried at 60 • C to weight constancy.
Other gr owth r equir ements (yeast extr act, peptone, CoM, acetate, and formate) were assessed after transferring the cultures at least three times in medium lacking a particular supplement, determining growth and methane production at 7 and 14 days after inoculation.A supplement was considered as essential if no gr owth occurr ed after 14 days or as stimulatory if growth and methane production after 14 days did not exceed that in full medium after 7 da ys .

Methane measurements
Aliquots of headspace gas (0.2 ml) were sampled every 7 days with a gas-tight syringe, and the methane content was analysed using a gas c hr omatogr a ph (SRI 8610C) equipped with a packed column (Por a pak Q, 80/100 mesh, 274 cm by 3.18 mm inside diameter) and a flame ionization detector.

Light and electron microscopy
For phase-contrast light microscopy of unfixed cells, an Axiophot photomicroscope (Zeiss, Oberkochen, Germany) was used.For photomicr ogr a phs, cultur es wer e a pplied to a gar-coated slides (Pfennig and Wagener 1986 ). Autofluorescence of cofactor F 420 was observed with the same microscope with a UV light source and a ppr opriate filter sets, using Methanobrevibacter ruminantium DSM 1093 as positive control.
Concentrated cell suspensions (3 μl) were high-pressure frozen, freeze-substituted with an acetone solution containing 0.25% osmium tetroxide, 0.2% uranyl acetate, and 5% H 2 O), and embedded in Epon 812 substitute resin (for details, see Renicke et al. 2017 ).Ultrathin sections (50 nm) were cut with a microtome equipped with a diamond blade.Sections were poststained with aqueous solutions of 2% ur an yl acetate and 0.5% lead citrate .T he sections were examined with a JEM-2100 transmission electron microscope (JEOL, Tok y o, J apan) operated at 120 kV.Images were acquired with a F214 fast-scan CCD camera (TVPIS, Gauting, Germany).

Genome sequencing and annotation
High molecular weight DNA from isolates was pr epar ed with the DNAEasy Blood & Tissue Kit (Qiagen) following the manufactur er's pr otocol.The pr ocedur es for genome sequencing, assembl y, and annotation hav e been pr e viousl y described (Pr otasov et al. 2023 ).Meta genome-assembled genomes wer e obtained fr om v arious species of termites and annotated in a pr e vious study (Hervé et al. 2020 ).All genomes and 16S rRNA genes are available at NCBI GenBank (accession numbers ar e giv en in the corresponding figures and in the Taxonomy section).For the analysis of the metabolic pathwa ys , annotation r esults wer e v erified, and missing functions were identified using Blast with a threshold E-value of 1E-5 and BlastKoala (Kanehisa et al. 2016 ).Amino acids biosynthesis pathw ays w er e additionall y c hec ked and annotated with GapMind (Price et al. 2020 ).

Phylogenetic analysis
The 16S rRNA gene sequences of the isolates and MAGs were imported into the Dictyopteran gut micr obiota r efer ence database (DictDb v. 5.1 Arc haea) (Pr otasov et al. 2023 ) and aligned against the existing alignment of pr e viousl y published sequences with the SINA aligner using the ARB software package (Ludwig et al. 2004, Pruesse et al. 2012 ).After manual curation of the alignment, a maxim um-likelihood tr ee was calculated using IQ-TREE 2 with the substitution model GTR + I + G4 (Kalyaanamoorthy et al. 2017, Minh et al. 2020 ).Node support was assessed using the Shimodair a-Hasegawa a ppr o ximate-lik elihood ratio test (Guindon et al. 2010, Hoang et al. 2018 ).
The genomes from isolates and MAGs were phylogenetically classified within the taxonomic fr ame work of the Genome Taxonomy Database (GTDB, release 207.2) using the GTDB toolkit (v.2.1.1)(Chaumeil et al. 2020, Hervé et al. 2020 ).A maximumlikelihood tree was inferred from a concatenated alignment of 53 ar chaeal single-cop y marker genes generated by GTDB toolkit using IQ-Tree with the substitution model LG + F + R4 selected by the ModelFinder tool and nonpar ametric bootstr a p br anc h support (Kalyaanamoorthy et al. 2017, Rinke et al. 2021 ).T he a v er a ge nucleotide identities of the genomes were calculated with FastANI version 1.3 (Jain et al. 2018 ).

Da ta visualiza tion and sta tistical anal ysis
Statistical analyses were performed with R v3.5.1, and plots were generated with ggplot2 (Wickham 2016 ).Genome size and number of protein coding genes were tested for normality of distribution with Sha pir o-Wilk normality test, Kruskal-Wallis, and post hoc Dunn tests with Holm P -value adjustment were performed using the ggbetweenstats function from the ggstatsplot package in R (Patil 2021 ).

Isolation and morphological characterization
Six strains of methyl-reducing Methanosarcinales were isolated fr om enric hment cultur es on H 2 + methanol that were inoculated with gut homogenates of the millipedes A. caudulanus (strain Ac7), A. gigas (strain Ag5), and A. monilicornis (strain Am2), and of the coc kr oac hes A. tessellata (strain At1), E. serranus (strain Es2), and H. flexivitta (strain Hf6).The isolates were obtained from single colonies in deep-agar dilution series; the dilution series were repeated to ensure the clonality of the culture (Pfennig and Trüper 1981 ).All strains formed lens-shaped, cream-colored colonies that r eac hed a diameter of up to 2 mm after 1 month of incubation ( Fig. A1 ).
Phase-contr ast micr oscopy of liquid cultur es in the earl y exponential phase sho w ed nonmotile, irregular cocci with a diameter of 1.0-2.0μm that resembled the cells of M. blatticola (Fig. 1 A-G).None of the strains formed cell clusters typical of Methanosarcina barkeri and some other Methanosarcina spp.(Fig. 1 H); only small clusters of cells were observed on rare occasions.All strains exhibited the c har acteristic fluor escence of cofactor F 420 in all growth phases, but it was weaker than in Methanosarcina spp.Ultrathin sections of strains Ag5 and Es2 revealed the presence of an Slayer surrounding the cytoplasmic membrane, but unlike M. barkeri , sho w ed no evidence for the formation of methanochondroitin (Fig. 2 ).
The isolates were not inhibited by kanamycin, ampicillin, or vancomycin (100 μg/ml each).Ho w ever, no gro wth occurred in the presence of chloramphenicol (10 μg/ml).

Di v ersity and phylogenomic analysis of the intestinal clade
Phylogenomic analysis of the Methanosarcinaceae family, including the metagenome-assembled genomes (MAGs) obtained in previous studies (Hervé et al. 2020, Protasov et al. 2023 ), confirmed the monophyletic origin of the r epr esentativ es fr om insects and  millipedes (Fig. 3 ).The three isolates from cockroach guts fell into the radiation of the genus Methanimicrococcus , which comprises a single cultured re presentati ve , M. blatticola.T he three isolates from millipedes formed a separate genus-level cluster, for which the name Methanolapillus is proposed (see the section "Taxonomy").They occupy a sister position to a clade that consists of MAGs from the digestive tract of mammals and an anaerobic digester inoculated with c hic ken feces (Campanar o et al. 2020, Xie et al. 2021 ), whose members have been assigned to the genus "Methanofrustulum " under SeqCode (Protasov et al. 2023 ).Together, the three genera form a monophyletic clade that consists exclusively of methanogens from intestinal tracts.
Also in 16S rRNA-based analyses, the strains from cockroaches fell into the radiation of the genus Methanimicrococcus , together with numerous uncultured re presentati ves from a wide range of coc kr oac hes and termites (Fig. 4 ).The str ains fr om millipedes formed a separate cluster that consists exclusiv el y of sequences deriv ed fr om div erse millipede species that were assigned to the genus Methanolapillus (Protasov et al. 2023 ).Both clusters occupy a sister position to uncultured methanogens from the digestive tr act of v arious mammals, including a fe w clones fr om permafr ost soil that may have originated from animal feces.Members of the genus "Methanofrustulum" were first detected in the cow rumen (Tajima et al. 2001, Wright et al. 2007 ) and subsequently in reindeer (Sundset et al. 2009 ), water buffalo (Chaudhary et al. 2012 ), yak (Huang et al. 2012 ), and sheep (Huang et al. 2016 ), and also in horse feces (Murru et al. 2018 ).The low abundance of "Methanofrustulum " in mammalian samples stands in sharp contrast to the situation in arthropod guts, where Methanimicrococcus or Methanolapillus may r epr esent up to 97% of the archaeal comm unity (Pr otasov et al. 2023 ).
The closest cultured relatives of intestinal Methanosarcinales are members of the genus Methanosarcina , which occur in soil, sediments, or anaerobic digesters (Wagner 2020 ).The lo w 16S rRN A gene sequence similarities (94.4%-96.0%)between the strains from millipedes (Ac7, Ag5, and Am2) and r epr esentativ es of Table 1.Phenotypic c har acteristics of the ne w isolates compar ed to the type species of the gener a Methanimicrococcus   (Müller et al. 1986 ).The presence of k e y genes of methanogenesis is indicated.1, Wood-Ljungdahl pathway; 2, methyl-reducing pathway; and 3, other ener gy-conv erting membrane complexes .T he m ultiunit complex is labeled as complete if at least half of the subunits pr esent.For mor e details, see Table A3 .

F m d /F w d F tr M c h M td M e r M tr A -H M c r F r h
the genus Methanimicrococcus and the av er a ge nucleotide identities of their genomes (ANI, 76%-78%) ( Fig. A2 ) support their placement in a separate genus (Yarza et al. 2014, Jain et al. 2018 ).

Energy metabolism
The members of the intestinal clade have estimated genome sizes ranging between 1.58 and 2.24 Mb, which is significantly smaller than the values of their closest r elativ es in the genus Methanosarcina (Table 1 ; Fig. 3 ; Fig. A3 ).
Compar ativ e genome anal ysis r e v ealed that in all cases, the reduction in genome size is accompanied by a loss of both catabolic and anabolic pathwa ys .T he most striking feature of the intestinal lineages is the absence of the Wood-Ljungdahl pathway and other prominent elements of the energy metabolism of their next relatives in the genus Methanosarcina .This documents that the se v er e genome r eduction pr e viousl y observ ed in M. blatticola is a common trend among all members of the entire intestinal clade and most likel y r eflects featur es that wer e pr esent alr eady in the last common ancestor of the three animal-associated lineages (Thomas et al. 2021 ) (Fig. 5 ).
The breakdown of the Wood-Ljungdahl pathway in all members of the intestinal clade explains their inability to reduce CO 2 for methane pr oduction, whic h had already been demonstrated for M. blatticola (Sprenger et al. 2005 ).The conservation of meth yl-tetrah ydromethanopterin (H 4 MPT): CoM methyl transferase (MtrA-H), and methylene-H 4 MPT reductase (Mer) in all genomes is most likely due to their role in anabolism, providing methyl-H 4 MPT and methylene-H 4 MPT as precursors for the biosynthesis of acetyl-CoA, methionine , thymidylate , and coenzyme A (Thauer 2012 ).Notabl y, methen yl-H 4 MPT cyclohydr olase (Mch) is absent from all members of the intestinal clade .T he absence of CdhA, CdhB (and CdhC in case of Methanolapillus ), which are k e y components of the CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex and essential for autotrophic growth, explains the acetate r equir ement of all str ains fr om the intestinal clade (Table 1 ).
All members of the intestinal clade lack the energy-conserving membrane-bound complexes typical of other Methanosarcinales , suc h as F 420 H 2 dehydr ogenase (Fpo), Rnf complex, and an ener gy-conv erting hydr ogenase (Ec h).An MtrA-H complex is present but it likely serves only in the methylation of H 4 MPT for anabolic purposes .T he only means for ener gy conserv ation during H 2 -dependent r eduction of methyl-CoM is the short electr on-tr ansport c hain formed by the methanophenazine-reducing [NiFe] hydrogenase (VhtACG) and the methanophenazine-oxidizing heterodisulfide reductase (HdrDE) (Fig. 5 ).All members of the intestinal clade possess sever al methyltr ansfer ase systems that transfer the methyl groups of methanol (Mta) and mono-, di-, and trimethylamine (Mtm, Mtb, and Mtt) to CoM for their subsequent reduction to methane (Fig. 3 ).They ar e or ganized in gene clusters that comprise a substrate-specific component, a methyl-accepting corrinoid protein (CoP), and in most cases also homologs of the second methyltr ansfer ase that tr ansfers the methyl gr oup fr om CoP to CoM (for details, see Table A3 ).In addition, all isolates encode homologs of a meth ylthiol: CoM meth yltr ansfer ase (Mts), whic h is involv ed in methanogenesis from dimethylsulfide and methylmerca ptopr opionate in M. barkeri (Tallant et al. 2001 ).In se v er al Methanimicrococcus and Methanolapillus genomes, we found homologs of an arsenite methyltr ansfer ase (ArsM).Homologs of the r ecentl y discov er ed methyltr ansfer ase of Methermicoccus shengliensis , which transfers methyl gr oups fr om methoxylated ar omatic compounds (Kurth et al. 2021 ), were not detected, which is consistent with the inability of the isolates to utilize such substrates.
Many members of the gen us Methanosar cina and other Methanosarcinales use glycogen and polyphosphates as storage compounds (Ferry 2012 , Wang et al. 2019 ).Genes involv ed in gl ycogen biosynthesis were absent from all re presentati ves of the intestinal clade, whereas the pathways for polyphosphate synthesis and degradation were present ( Table A3 ).

Anabolism
Compar ativ e genome anal ysis r e v ealed that most of the intestinal Methanosarcinales lack the biosynthetic pathways for aromatic amino acids phen ylalanine, tyr osine, and tryptophan with some Methanimicrococcus likely also not able to synthesize valine, leucine, and isoleucine ( Table A3 ).In contrast to members of the gen us Methanosar cina , whic h ar e typicall y ca pable of dinitr ogen fixation, none of the genomes of the intestinal clade encoded homologs of nitrogenase (NifDHK).
All members of the intestinal clade lack the biosynthetic pathway for methanofuran ( Table A3 ) (Wang et al. 2015 ).Only Methanimicrococcus strains Es2 and Hf6 retained the l -phosphoserinedependent pathway for the biosynthesis of CoM, which is common among Methanosarcinales (Graham et al. 2009 ).The absence of the pathway from other Methanimicrococcus spp., including M. blatticola , and all members of the genus Methanolapillus , explains their r equir ement for CoM as gr owth factor (Table 1 ).Genes encoding the PEP-dependent pathway or the bacterial pathway for CoM biosynthesis were not found.
While most genomes encode the complete biosynthetic pathways for pantothenate, niacinamide, and thiamine, the pathways for the synthesis of se v er al other cofactors are absent or incomplete ( Table A3 ).Most isolates should have a requirement for folate and (except for the genus Methanolapillus ) pyridoxalphosphate .T he pathwa y for biotin synthesis is absent but compensated by an ABC transporter for biotin.Thiamine cannot be synthesized de novo but via a salv a ge pathway requiring some precursors from the environment (Jenkins et al. 2007 ) 3 and Table A3 .MTS, methyl tr ansfer ase systems; MP, methanophenazine.
medium, it is likely that the pathway for riboflavin biosynthesis in Methanosarcinales differs from the canonical pathway.
T he pathwa ys for the biosynthesis of tetr a pyrr oles ar e full y pr eserv ed in all genomes of the intestinal clade ( Table A3 ).They comprise both the initial steps common to all tetr a pyrr oles and the upper parts of the pathways leading to cofactor F 430 , cobalamine/cobamides and heme (Bryant et al. 2020 ).

Cell envelope and reactive oxygen species
All genomes encode homologs of the protein MA0829, which selfassembles into the two-dimensional crystalline array forming the S-layer of Methanosarcina acetivorans (Francoleon et al. 2009, Arbing et al. 2012 ).Although the identity scor es ar e r ather low (28%-36%; Table A3 ), this is consistent with the a ppar ent pr esence of an S-layer in the electr on micr ogr a phs of Methanimicrococcus and Methanolapillus spp.(Fig. 2 ).
The nonmotile phenotype of the isolates is consistent with the absence of the arc haellum oper on (Arl) and the chemotaxis operon (Che) present in some Methanosarcina (Jarrell et al. 2021 ) from all members of the intestinal clade.Homologs of the subcluster ArlIJ, which were detected in a few genomes, are likely involv ed in pr otein export, as pr edicted alr ead y for M. mazei (De ppenmeier et al. 2002 ).
All members of the intestinal clade encode catalase, thioredo xin-de pendent pero xiredo xins, and supero xide reductase .While Cu-F e super oxide dism utase and F 420 H 2 oxidase are absent, a Fe-Mn superoxide dismutase (SodA) was detected in members of the genus Methanimicrococcus ( Table A3 ).Notably, the homolog of cytoc hr ome bd (CydAB), which is encoded by many members of the genus Methanosarcina (Baughn andMalamy 2004 , Br oc hier-Armanet et al. 2009 ), is conserved in all members of the intestinal clade (Fig. 3 ).Phylogenetic analysis revealed a close relationship to the high-affinity quinol oxidases of Bacillota (Fig. 7 ).

Genome reduction in the intestinal clade
While members of the genus Methanosarcina have experienced a massive genome expansion due to horizontal gene transfer from bacteria and possess the largest genomes in the archaeal domain (Deppenmeier et al. 2002, Garushyants et al. 2015 ), members of the intestinal clade (genera Methanimicrococcus , Methanolapillus , and "Methanofrustulum ") show a significant genome reduction (Fig. 6 ).Small genomes with an av er a ge size of 2.0 Mb are also found among Methanosarcinaceae from hypersaline environments (genera Methanohalobium , Methanohalophilus , and Methanosalsum ) (Oren 2014a ).While genome reduction in intestinal micr oor ganisms has been explained with the loss of nonessential genes in nutrientric h envir onments (Morris et al. 2012 ), genome str eamlining in hypersaline habitats may be attributed to a strong purifying selection driven by the extremely nutrient-poor environment (Wolf andK oonin 2013 , Gio v annoni et al. 2014 ).Notabl y, the av er a ge number of protein-encoding genes and the corresponding average coding density in the intestinal clade of Methanosarcinales is significantly lo w er than in those fr om hypersaline envir onments (Fig. 6 ; Figure A4 ), suggesting that numerous genes were pseudogenized but not yet eliminated from the genome due to the absence of a strong purifying selection (Koonin and Wolf 2008 ).This underscores that the environment is an important driver of genome evolution in Methanosarcinales .
The loss of the Wood-Ljungdahl pathway and the absence of many energy-converting membrane complexes present in the genus Methanosarcina (Fpo, Ech, and Rnf) is common to all  and A2.
members of the intestinal clade (Fig. 3 ).As a consequence, members of the intestinal clade can conserve energy only via the methanophenazine-linked electron transport chain that is formed by the complexes VhtACG and HdrDE (Abken et al. 1998 ).
Mor eov er, in the absence of Ech and Rnf, the reduced ferredoxin r equir ed for anabolism must be provided by other means.
A MvhAGD [NiFe] hydrogenase is generally absent from the order Methanosarcinales (Mand and Metcalf 2019 ), precluding the formation of reduced ferredoxin via a cytosolic MvhAGD-HdrABC complex.Since members of the intestinal clade possess both formate dehydrogenase (FdhAB) and an MvhD homolog fused with HdrABC, an alternativ e r oute would be the r eduction of ferr edoxin with formate, using an FdhAB-MvhD-HdrABC complex, as shown for Methanococcus maripaludis ( Methanococcales ) and Methanoculleus thermophilus ( Methanomicrobiales ) (Costa et al. 2010, Milton et al. 2018, Abdul Halim et al. 2021 ).An alternative electron donor for this complex would be F 420 H 2 , as suggested already by Thomas et al. ( 2021 ) for M. blatticola .The cytoplasmic HdrABC present in members of the intestinal clade belongs to the same subclade as its homolog in M. acetivorans (HdrA2B2C2), which couples F 420 H 2 oxidation to the sim ultaneous r eduction of ferr edoxin and CoM-S-S-CoB via flavin-based electron bifurcation (Yan et al. 2017 ).A structur al and mec hanistic anal ysis of the FdhAB-MvhD-HdrABC complex in Methanospirillum hungatei ( Methanomicrobiales ) r e v ealed that it uses both formate and F 420 H 2 as electron donor (Watanabe et al. 2021 ).Since we observed a growth requirement for formate only in strain Am2, it is likely that members of the intestinal clade do not use formate for ferr edoxin r eduction but instead use F 420 H 2 generated by the F 420 -reducing [NiFe] hydrogenase (Frh) (Fig. 5 ).
While MtrA-H is the only site of energy conservation in hydr ogenotr ophic, CO 2 -r educing methanogens, members of Methanosarcinales possess additional ion-translocating membr ane-bound complexes, whic h incr eases the ATP yield per mol of methane and explains why their growth yields are the highest among methanogens (Thauer et al. 2008 , Mand andMetcalf 2019 ).Growth yields among members of the intestinal clade (3.0-5.0 g/mol methane; Table 1 ) are in the same range as those of M. barkeri grown on methanol + H 2 with acetate as carbon source (4.6 g/mol methane) (Müller et al. 1986 ) but substantially lo w er than those of M. barkeri grown only on methanol (7.2 g/mol) (Weimer and Zeikus 1978 ).A similar growth yield (4 g/mol methane) was reported for the obligately meth yl-reducing Methanosphaer a stadtmanae (Miller and Wolin 1985 ), which oxidizes the reduced Fd produced by HdrABC with an ener gy-conv erting hydr ogenase (Ehb (Thauer et al. 2008 )).
In this context, it is important to note that the fr ee-ener gy changes of the methyl-reducing pathway (Eq. 1 ) and the methyldisproportionating pathway (Eq. 2 ) are almost identical under standard conditions, whereas the hydrogen-dependent reduction of methanol to methane becomes ener geticall y less favor able at lo w er hydrogen partial pressure (Thauer et al. 2008, Feldewert et al. 2020 ).

CH
(2) The lo w er gro wth yield of the obligatel y methyl-r educing Methanomassiliicoccus luminyensis (2.4 g/mol methane) (Kröninger et al. 2017 ) is explained by the unique energy metabolism of Methanomassiliicoccales , wher e onl y e v ery second CoM-S-S-CoB is reduced via an ener gy-conv erting membr ane-bound complex (Lang et al. 2015, Kröninger et al. 2019 ).

Evolution of the methylotrophic metabolism
The intestinal Methanosarcinales clade r epr esents a case of obligate H 2 -dependent meth ylotroph y.They utilize a short electr on tr ansport c hain that consists of onl y membr ane-bound heter odisulfide r eductase HdrDE and membr ane-bound VhtACG hydrogenase .T he intestinal clade is embedded among Methanosarcinales that have the capacity for h ydrogenotrophic, meth yldisproportionating, and aceticlastic methanogenesis .T he transition to obligate H 2 -dependent methyl-reducing methanogenesis was likely triggered by the dispersal of an ancestral lineage of Methanosarcinales into the H 2 -rich gut environment.Since methanol oxidation in M. barkeri is inhibited in the presence of H 2 (Mand et al. 2018 ), it is possible that this restricted the intestinal clade to using only the methyl-reducing pathway.Once the selective pressure on methyl oxidation had been r emov ed, members of the clade lost the Wood-Ljungdahl pathway and the membrane-bound complexes involved in methyl oxidation and became obligately H 2 -dependent methylotrophs .T he loss of the Wood-Ljungdahl pathway also lead members of the clade to r el y on the gut envir onment for pr ecursors of biosynthesis .T hat would also explain why intestinal Methanosarcinales are r ar el y detected in nongut environments (Thomas et al. 2022 ).The oligotrophic conditions in sediments and soils, where other Methanosarcinales are typically encountered, does not meet the gr owths r equir ement of the auxotr ophic intestinal species.Rar e observations of Methanimicrococcus -related sequences in soil (see Fig. 4 ) may indicate fecal contaminations from mammals and/or arthropods.
Ho w e v er, they all differ in the way they produce an ion gradient for ATP production and do not use the MtrA-H complex that is common for other methanogens (summarized in Garcia et al. 2022 ).The differences in the pathways indicate an independent origin of meth ylotroph y in these lineages .Moreo ver, it has been suggested that also se v er al other lineages of methanogens ar e obligatel y methylotr ophic, based either on compar ativ e genome analysis alone (Nobu et al. 2016, Vanwonterghem et al. 2016 ) or in combination with a physiological c har acterization of isolates and enrichment cultures (Kohtz et al. 2024, Krukenberg et al. 2024, Lynes et al. 2024, Wu et al. 2024 ).The differences between these lineages suggest a convergent evolution of the obligate methyl-reducing pathwa ys , starting with a unique biochemical basis in different lineages.Evidently, the gut environment stands out as a significant hub for obligate methyl-reducing methanogens harboring re presentati ves such as Methanosphaera spp., Methanomethylophilaceae , and intestinal Methanosarcinales .This implies that the gut environment not only supports but potentially even facilitates the transition to obligate H 2 -dependent methyl-reducing methanogenesis, as shown for intestinal Methanosarcinales in the present study.

The loss of biosynthetic capacities in the intestinal clade
All strains of intestinal Methanosarcinales have pronounced auxotr ophic phenotypes, r equiring gr owth factors suc h as acetate, formate, and/or other, so far unidentified components of yeast extr act.A r equir ement for CoM is common not only among repr esentativ es of the genera Methanimicrococcus and Methanolapillus (Sprenger et al. 2000 , Table 1 ) but also among gut-associated lineages of Methanobacteriales ( Methanobrevibacter spp., M. stadtmanae ) and Methanomassiliicoccales ( Ca .Methanoplasma termitum) (Oren 2014b , Lang et al. 2015 ).In arthropod guts, members of the intestinal clade always co-occur with other methanogens (Protasov et al. 2023 ), suggesting a dependency of these species on other, CoM-producing methanogens in the intestinal community.
The absence of a functional CODH/ACS complex and presence of an acetate transporter (Welte et al. 2014, Ribas et al. 2019 ) explains the acetate r equir ement of all isolates in the genera Methanimicrococcus and Methanolapillus and predicts the same also for all uncultured members of the intestinal clade.It is intriguing that Methanimicrococcus and Methanolapillus differ in the number of Cdh subunits; Methanimicrococcus have only two out of five subunits, while Methanimicrococcus possess three subunits ( Table A3 ).Like CoM auxotrophy, also a growth requirement for acetate is common among methanogens isolated from intestinal en vironments , such as Methanobrevibacter or Methanosphaera spp.(Oren 2014b ).It is likely that the loss of acetate production via the CODH/ACS complex is promoted by the high acetate concentration in intestinal en vironments .
Tetr a pyrr oles ar e essential components of man y enzymes and cofactors of methanogens, such as cofactor F 430 (the prosthetic group of methyl-CoM reductase), cobalamin/cobamide (the prosthetic group of methyltransferases), and heme (the prosthetic group of cytochromes) (Matthews et al. 2008, Bryant et al. 2020 ).While cofactor F 430 is unique to methanogens and is synthesized by all species isolated to date, cobalamin/cobamide are produced by many bacteria and can be scavenged from the envir onment (Sok olo vska ya et al. 2020 ).The presence of the complete pathway for the synthesis of cobalamin and the lack of a B 12 transporter in all isolates of the intestinal clade ( Table A3 ) indicates the importance of B 12 -dependent methyltr ansfer ases in their energy metabolism.Also, the biosynthetic pathway for pyrr ol ysine, an essential amino acid in methylamine methyltransferases, in almost all genomes ( Table A3 ) is in agreement with the ability to grow on methylamines (Table 1 ) (Rother and Krzycki 2010 ).
Heme can be synthesized by all methanogens that produce cytoc hr omes ( Methanosarcinales and Methanonatronarchaeales ; Mand andMetcalf 2019 , Steiniger et al. 2022 ) and possibly a few uncultur ed linea ges (Ou et al. 2022 ).Lik e other archaea, Methanosar cinales synthesize heme via the siroheme pathway (Dailey et al. 2017 ).The presence of the pathway in all members of the intestinal clade and the absence of a heme transporter reflects the importance of cytoc hr ome-containing complexes (VhtACG and HdrDE) in their energy metabolism.
Amino acid and vitamin auxotrophies are common for complex micr obial comm unities suc h as gut micr obiota (Zengler andZaramela 2018 , Ramoneda et al. 2023 ).While many Methanosarcina species from soils and sediments grow on medium without organic growth factors (Wagner 2020 ), members of the intestinal clade ar e auxotr ophic for se v er al amino acids and vitamins ( Table A3 ).Again, the same applies to other host-associated archaea, such as Methanobrevibacter spp.(Leadbetter and Breznak 1996 ) and M. stadtmanae (Miller and Wolin 1985 ).
The intestinal clade members do not possess the genes for the synthesis and activation of N -acetyl-galactosamine and glucuronic acid, the precursors for methanochondroitin (Hartmann and König 1991 ), as already shown for M. blatticola (Thomas et al. 2021 ).This is consistent with absence of a ggr egates in all isolates from the intestinal clade under tested growth conditions and the a ppar ent absence of methanoc hondr oitin fr om their cell envelope (Fig. 2 ).Ho w ever, like most methanogens, members of Methanosarcinales possess a proteinaceous S-layer that is linked to the cytoplasmic membr ane (v an Wolfer en et al. 2022 ).Homologs of the S-layer protein were present in all members of the intestinal clade, and an S-lay er w as visualized in two of the isolates.
Although members of the genus Methanosarcina are typically immotile, M. acetivorans possesses genes r equir ed for the synthesis of an arc haeal fla gellum (arc haellum) and c hemotaxis (Wagner 2020 ).All isolates of the genera Methanimicrococcus and Methanolapillus are immotile and lack the genes for an archaellum or c hemotaxis.Epifluor escence micr oscop y of the gut w all of cockr oac hes sho w ed cells r esembling Methanimicrococcus spp.attac hed to the inner surface of the gut epithelium (Sprenger et al. 2000 ).It is likely that an attachment to intestinal surfaces is a general strategy for all members of the intestinal clade and led to a loss of both motility and chemical sensing upon transition to the gut envir onment, as alr eady suggested for M. blatticola (Thomas et al. 2021 ).

Relationship to oxygen
Although oxygen penetrates into the hindgut of arthropods and renders the gut periphery a microoxic habitat, the inner surface of the hindgut wall is often colonized by methanogens (Brune 2019 ).It is long known that both Methanobrevibacter and Methanisarcina species are more oxygen tolerant than other methanogens (Kiener and Leisinger 1983 ).Experiments with a gar-gr adient tubes and dense cell suspensions documented that arthr opod-deriv ed members of the genus Methanobrevibacter and M. blatticola remov e O 2 fr om their envir onment (Leadbetter and Br eznak 1996, Sprenger et al. 2007, Tholen et al. 2007 ).Methanobrevibacter species reduce O 2 to water via an F 420 H 2 oxidase (FprA) (Seedorf et al. 2004 ).Lik e other Methanosar cinales , members of the intestinal clade encode enzymes for the detoxification of r eactiv e oxygen species, such as catalase (Shima et al. 1999 ), superoxide dismutase (Brioukhanov et al. 2000 ), and super oxide r eductase (Krätzer et al. 2011 ), and a thioredoxin system (McCarver andLessner 2014 , Kumar et al. 2015 ).Homologs of FprA, ho w e v er, ar e absent ( Table A3 ).
A plausible candidate for O 2 r emov al in the intestinal clade is cytoc hr ome bd (CydAB), whic h is pr esent in the genomes of many Methanosarcinaceae and is most closely related to the highaffinity quinol oxidases of Bacillota (Fig. 7 ).Homologs of this enzyme allow "obligate anaer obes" suc h as Bacteroides fragilis or Desulfovibrio gigas to r emov e O 2 fr om their immediate envir onment and e v en support a r espir atory ener gy metabolism under microoxic conditions, which gave rise to the concept of "nanaerobes" (Lemos et al. 2001 , Baughn andMalamy 2004 ).We propose that the CydAB of Methanosarcinales is part of a methanophenazine-linked r espir atory c hain (Fig. 5 ).Analogous to the hydr ogen-dependent r eduction of heter odisulfide via VhtACG-methanophenazine-HdrDE, whic h pr oduces an electr oc hemical pr oton gr adient during methanogenesis via a mec ha-nism similar to that of the bacterial quinone loop (Deppenmeier 2004 ), also the hydrogen-dependent reduction of O 2 via VhtACGmethanophenazine-CydAB may serve to conserve energy by electr on tr ansport phosphorylation.

Taxonomy
Recentl y, se v er al members of the intestinal Methanosarcinales , including the isolates in the pr esent study, hav e been described as new species under SeqCode, using their genomes as type material (Protasov et al. 2023 ).Ho w ever, taxa described under SeqCode ar e not consider ed v alidl y published under the ICNP (Göker et al. 2022 ) because the latter gener all y r equir es axenic cultur es to be deposited as type material in two culture collections .Here , we provide a formal description of the six isolates and the new genus Methanolapillus under the ICNP, following also minimal standards for the description of methanogens (Prakash et al. 2023 ).
Description: Cells are irregular cocci with a diameter of 1.0-2.0μm, occurring singly or sometimes in small clusters.Form lens-sha ped, cr eam-color ed colonies in deep-a gar cultures .Nonmotile .Obligately hydrogen-dependent methylr educing methanogens; r educe methanol and methylamines, but not methoxylated aromatic compounds.No methane formation from formate , acetate , or ethanol.Mesophilic.Require complex medium with acetate and Casamino acids or peptone.Yeast extract and formate may be stimulatory or essential for growth.

Figure 3 .
Figure3.Phylogenomic tree illustrating the relationship of isolates and MAGs from animal guts to members of the genus Methanosarcina , and important features of their respective genomes.Newly proposed species are shown in bold.The tree is based on a concatenated alignment of 53 markers and was reconstructed with IQ-Tree under the LG + F + I + G4 model of evolution.All nodes are fully supported ( > 99%).The tree was rooted using other Methanosarcinaceae as outgroup.Genome size of MAGs was estimated based on assembly size and completeness (determined with CheckM).The presence of k e y genes of methanogenesis is indicated.1, Wood-Ljungdahl pathway; 2, methyl-reducing pathway; and 3, other ener gy-conv erting membrane complexes .T he m ultiunit complex is labeled as complete if at least half of the subunits pr esent.For mor e details, see TableA3.

Figure 4 .
Figure 4. Phylogenetic 16S rRNA tree illustrating the relationship of strains isolated from arthropods to other re presentati ves of the family Methanosarcinaceae .Ne wl y pr oposed and type species ar e shown in bold.The maxim um-likelihood tr ee is based on a cur ated alignment of near-full-length 16S rRNA genes ( > 1400 sites) and was generated using IQ-TREE under the GTR + I + G4 model of evolution.Node support was tested with ultrafast bootstrap analysis ( •≥95% and • ≥70%; 1000 replicates).The scale bar indicates the number of substitutions per site.Color coding indicates host groups.